This study examines the effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)(2)D(3) or its derivatives, but significantly decreased in the presence of the two retinoids (0.001--10 microM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060, and 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 microM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 microM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)(2)D(3) or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 microM 9-cis retinoic acid and 10 nM 1,25(OH)(2)D(3) or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.